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Topic:Semen Preservation
Semen preservation involves the collection, processing, and storage of stallion semen for future use in artificial insemination. This practice enables the extension of genetic material across geographical boundaries and temporal constraints. The preservation process typically includes semen evaluation, dilution with extenders, cooling, and sometimes cryopreservation. Factors such as semen quality, extender composition, and storage conditions influence the success of preservation. This page compiles peer-reviewed research studies and scholarly articles that explore techniques, challenges, and advancements in the field of equine semen preservation, focusing on optimizing fertility outcomes and extending the reproductive lifespan of stallions.
Enzymatic scavengers in the epididymal fluid: comparison between pony and miniature breed stallions. The use of stallion semen collected from cauda epididymis for AI has increased due to the new protocols available for cryopreservation. Preserving the genetic material from valuable males that suffer sudden death or other events that prematurely end the stallion's reproductive life is an important strategy for Stud breeding management. While protecting spermatozoa from oxidative stress and infectious agents, the epididymis promotes the enhancement of sperm cell morphology and changes in membrane protein profile, increasing its fertility potential. The epididymal fluid must be a balanced redox ...
Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5′ AMP-activated protein kinase (AMPK). This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization ...
Localization and functional modification of L-type voltage-gated calcium channels in equine spermatozoa from fresh and frozen semen. It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level fo...
Effect of overnight staining on the quality of flow cytometric sorted stallion sperm: comparison with tradtitional protocols. Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sort...
Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus. Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Current status of freeze-drying technology to preserve domestic animals sperm. In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics. Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen's microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorgan...
Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen. Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specifici...
Comparison of the Effect of Heterologous and Homologous Seminal Plasma on Motility and Chromatin Integrity of Stallion Spermatozoa Selected by Single Layer Centrifugation. The effect on sperm motility and chromatin integrity of adding homologous or heterologous equine seminal plasma (SP) to fresh stallion spermatozoa selected by single layer centrifugation (SLC) was studied. No statistical difference in mean progressive motility was seen after adding SP at time 0 h, although there were differences for individual stallions. The proportion of spermatozoa with high velocity was increased compared to untreated SLC-selected spermatozoa (P < 0.05), with significant differences between individuals (P < 0.01). When the SLC samples were stored for 24 h before a...
Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions? The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n = 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were in...
Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96® extender. The rise of assisted reproduction techniques in equine medicine has fostered investigations that seek to optimize methods to increase fertility rates. Since cryopreservation continues to give low values of viability in stallions, the handling and preservation of the sperm is of vital importance. This reduction of fertility makes it essential for farmers to find new options that ensure reliability in the use of these techniques. The main objective of this study was to assess the effect of INRA 96® (manufactured commercial extender for cooling of Equine semen) as an extender for cryopreservatio...
Caspase activation, hydrogen peroxide production and Akt dephosphorylation occur during stallion sperm senescence. To investigate the mechanisms inducing sperm death after ejaculation, stallion ejaculates were incubated in BWW media during 6 h at 37°C. At the beginning of the incubation period and after 1, 2, 4 and 6 h sperm motility and kinematics (CASA), mitochondrial membrane potential and membrane permeability and integrity were evaluated (flow cytometry). Also, at the same time intervals, active caspase 3, hydrogen peroxide, superoxide anion (flow cytometry) and Akt phosphorylation (flow cytometry) were evaluated. Major decreases in sperm function occurred after 6 h of incubation, although after 1 h ...
Naturally and stimulated levels of reactive oxygen species in cooled stallion semen destined for artificial insemination. The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with obse...
Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, w...
Effect of artificial insemination protocol and dose of frozen/thawed stallion semen on pregnancy results in mares. Deep intra-uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra-uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen-thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi-flexible Minitube pipette was compared with a newly de...
Effect of heterologous and homologous seminal plasma on stallion sperm quality. Removing most of the seminal plasma (SP) from stallion semen has been shown to improve survival during cooled storage, yet adding small quantities of SP may improve pregnancy rates or cryosurvival. Furthermore, there is considerable controversy about whether the stallion's own SP or heterologous SP produces the best effect, possibly because of the variation between stallions in SP proteins or because some homologous SP remained in the sperm preparation. The SP is removed completely from stallion spermatozoa prepared by colloid centrifugation. Thus, the aim of the present study was (1) to inves...
In vitro equine embryo production using air-dried spermatozoa, with different activation protocols and culture systems. The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P 0.05). Blastocysts were obtained with cooled spermatozoa, and ...
Influence of the uterine inflammatory response after insemination with frozen-thawed semen on serum concentrations of acute phase proteins in mares. The aim of this study was to investigate the clinical relevance of measuring blood concentrations of serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fib) in horse reproductive management, and changes in response to artificial insemination (AI) with frozen-thawed semen. Standardbred mares (n=18) with different reproductive status (eight healthy mares in first postpartum oestrus, five healthy barren mares and five mares with endometritis) were inseminated with frozen-thawed semen. Endometritis was evaluated during oestrus by bacteriological culture, cytology and presence of ultrasonicall...
Effect of extender and amino acid supplementation on sperm quality of cooled-preserved Andalusian donkey (Equus asinus) spermatozoa. The main aim of this study was to evaluate the efficacy of two commercially available liquid stallion semen extenders for the preservation of Andalusian donkey semen at 5°C for up to 72h, and to evaluate the effect of amino acid addition on sperm quality of cooled donkey semen. In addition, this study investigated the effect of seasons on semen characteristics of Andalusian jackasses. Throughout a year, 50 ejaculates were collected from ten adult donkeys and a complete semen evaluation was performed immediately after collection. In Experiment 1, semen samples (n=32) were pooled, divided into ...
Sperm abnormalities in post-thawed semen of Tunisian Arab stallions. The study was undertaken in order to evaluate sperm morphology features of post-thawed semen of Tunisian Arab stallions. Forty two ejaculates was collected and frozen, during years 2009 and 2010, from 9 stallions aged between 9 to 24 years. After thawing, sperm morphology was studied after eosin-nigrosin stain. The percentages of abnormal head, mid piece, flagella, sperm with droplets and the total abnormal sperm were determined. Analysis of variance was carried out using SAS software. The results showed that all sperm morphology features varied among ejaculates within stallion and among stall...
Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii). Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski's horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski's horse spermatozoa during liquid storage at 4°C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski's versus domestic horse. When Przewalski's horse spermatozoa were incubated at 4°...
Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm. An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples ...
Concentration, activity and biochemical characterization of myeloperoxidase in fresh and post-thaw equine semen and their implication on freezability. Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extr...
Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter. Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled-stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO...
Identification of apoptotic bodies in equine semen. Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and ...
Removal of bacteria from stallion semen by colloid centrifugation. Bacteria (environmental contaminants and occasionally potential pathogens) are found in most stallion ejaculates and may negatively affect sperm quality during storage. Since the use of antibiotics can lead to the development of resistance, an alternative means of microbial control is desirable. The removal of bacteria from stallion semen using Single Layer Centrifugation through Androcoll-E was investigated. Known doses of cultured bacteria were added to freshly collected ejaculates (15mL aliquots) before processing by Single Layer Centrifugation. The resulting sperm pellets and controls (not...
During cooled storage the extender influences processed autophagy marker light chain 3 (LC3B) of stallion spermatozoa. To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the ...
Cryopreservation of epididymal stallion sperm. Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion's breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen-thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was test...
Effect of Cholesterol and Equex-STM Addition to an Egg Yolk Extender on Pure Spanish Stallion Cryopreserved Sperm. Cholesterol and Equex-STM are frequently added to different commercial and experimental extenders improving postthawing sperm quality. Doses of 125-150 mM of cholesterol from pig liver and 0.5-0.7% of Equex-STM were evaluated in a standard eggyolk extender (Martin et al., 1979). Six ejaculates per stallion from six pure Spanish stallions (6-8 years old) were collected in Martin's extender (B) and different mixtures of 125 mM-0.5% (I), 125 mM-0.7% (II), 150 mM-0.5% (III), and 150 mM-0.7% (IV) were added to original Martin's extender. Samples were frozen in 0.5 mL straws (100 × 10(6...
Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses. The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Si...
